Microbiota-targeted therapies in inflammation resolution.

Gut microbiota has been shown to systemically shape the immunological landscape, modulate homeostasis and play a role in both health and disease. Dysbiosis of gut microbiota promotes inflammation and contributes to the pathogenesis of several major disorders in gastrointestinal tract, metabolic, neurological and respiratory diseases. Much effort is now focused on understanding host-microbes interactions and new microbiota-targeted therapies are deeply investigated as a means to restore health or prevent disease. This review details the immunoregulatory role of the gut microbiota in health and disease and discusses the most recent strategies in manipulating individual patient's microbiota for the management and prevention of inflammatory conditions.

Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus.

The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire.

The first international Rome consensus conference on gut microbiota and faecal microbiota transplantation in inflammatory bowel disease.

BACKGROUND: Several randomised clinical trials (RCTs) performing faecal microbiota transplantation (FMT) for the management of inflammatory bowel disease (IBD), particularly for ulcerative colitis, have recently been published, but with major variations in study design. These include differences in administered dose, route and frequency of delivery, type of placebo and evaluated endpoints. Although the overall outcomes appear to be promising, they are highly dependent on both donor and recipient factors. OBJECTIVE: To develop concensus-based statements and recommendations for the evaluation, management and potential treatment of IBD using FMT in order to move towards standardised practices. DESIGN: An international panel of experts convened several times to generate evidence-based guidelines by performing a deep evaluation of currently available and/or published data. Twenty-five experts in IBD, immunology and microbiology collaborated in different working groups to provide statements on the following key issues related to FMT in IBD: (A) pathogenesis and rationale, (B) donor selection and biobanking, (C) FMT practices and (D) consideration of future studies and perspectives. Statements were evaluated and voted on by all members using an electronic Delphi process, culminating in a plenary consensus conference and generation of proposed guidelines. RESULTS AND CONCLUSIONS: Our group has provided specific statements and recommendations, based on best available evidence, with the end goal of providing guidance and general criteria required to promote FMT as a recognised strategy for the treatment of IBD.

Loss of gut barrier integrity triggers activation of islet-reactive T cells and autoimmune diabetes.

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in "at-risk" individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications.

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.

License to Kill: When iNKT Cells Are Granted the Use of Lethal Cytotoxicity.

Invariant Natural Killer T (iNKT) cells are a non-conventional, innate-like, T cell population that recognize lipid antigens presented by the cluster of differentiation (CD)1d molecule. Although iNKT cells are mostly known for mediating several immune responses due to their massive and diverse cytokine release, these cells also work as effectors in various contexts thanks to their cytotoxic potential. In this Review, we focused on iNKT cell cytotoxicity; we provide an overview of iNKT cell subsets, their activation cues, the mechanisms of iNKT cell cytotoxicity, the specific roles and outcomes of this activity in various contexts, and how iNKT killing functions are currently activated in cancer immunotherapies. Finally, we discuss the future perspectives for the better understanding and potential uses of iNKT cell killing functions in tumor immunosurveillance.

Tumor Infiltrating Regulatory T Cells in Sporadic and Colitis-Associated Colorectal Cancer: The Red Little Riding Hood and the Wolf.

Regulatory T cells represent a class of specialized T lymphocytes that suppress unwanted immune responses and size the activation of the immune system whereby limiting collateral damages in tissues involved by inflammation. In cancer, the accumulation of Tregs is generally associated with poor prognosis. Many lines of evidence indicate that Tregs accumulation in the tumor microenvironment (TME) suppresses the immune response against tumor-associated antigens (TAA), thus promoting tumor progression in non-small cell lung carcinoma (NSLC), breast carcinoma and melanoma. In colorectal cancer (CRC) the effect of Tregs accumulation is debated. Some reports describe the association of high number of Tregs in CRC stroma with a better prognosis while others failed to find any association. These discordant results stem from the heterogeneity of the immune environment generated in CRC in which anticancer immune response may coexists with tumor promoting inflammation. Moreover, different subsets of Tregs have been identified that may exert different effects on cancer progression depending on tumor stage and their location within the tumor mass. Finally, Tregs phenotypic plasticity may be induced by cytokines released in the TME by dysplastic and other tumor-infiltrating cells thus affecting their functional role in the tumor. Here, we reviewed the recent literature about the role of Tregs in CRC and in colitis-associated colorectal cancer (CAC), where inflammation is the main driver of tumor initiation and progression. We tried to explain when and how Tregs can be considered to be the "good" or the "bad" in the colon carcinogenesis process on the basis of the available data concluding that the final effect of Tregs on sporadic CRC and CAC depends on their localization within the tumor, the subtype of Tregs involved and their phenotypic plasticity.

Gut Microbiota Manipulation as a Tool for Colorectal Cancer Management: Recent Advances in Its Use for Therapeutic Purposes.

Colorectal cancer (CRC) is a multifaceted disease influenced by both environmental and genetic factors. A large body of literature has demonstrated the role of gut microbes in promoting inflammatory responses, creating a suitable microenvironment for the development of skewed interactions between the host and the gut microbiota and cancer initiation. Even if surgery is the primary therapeutic strategy, patients with advanced disease or cancer recurrence after surgery remain difficult to cure. Therefore, the gut microbiota has been proposed as a novel therapeutic target in light of recent promising data in which it seems to modulate the response to cancer immunotherapy. The use of microbe-targeted therapies, including antibiotics, prebiotics, live biotherapeutics, and fecal microbiota transplantation, is therefore considered to support current therapies in CRC management. In this review, we will discuss the importance of host-microbe interactions in CRC and how promoting homeostatic immune responses through microbe-targeted therapies may be useful in preventing/treating CRC development.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

BACKGROUND: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (TR1) cells but is also produced by CD25+ regulatory T (Treg) cells. OBJECTIVE: We aimed to identify and characterize human intestinal TR1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). METHODS: CD4+ T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4+ T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1ß and IL-23 responsiveness was assessed. RESULTS: Intestinal TR1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5+PD-1+ TR1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ+ TR1 cells, but not IL-7 receptor-positive TH cells or CD25+ Treg cells, showed lower IL-10 expression in patients with IBDs. TR1 cells were responsive to IL-23, and IFN-γ+ TR1 cells downregulated IL-10 with IL-1ß and IL-23. Conversely, CD25+ Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. CONCLUSIONS: We provide the first ex vivo characterization of human intestinal TR1 cells. Selective downregulation of IL-10 by IFN-γ+ TR1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

Absence of a role for interleukin-13 in inflammatory bowel disease.

IL-13 has been implicated in the pathogenesis of ulcerative colitis (UC), and may have a role in animal models of gut fibrosis. We studied the involvement of IL-13 in inflammation and fibrosis in UC and Crohn's disease (CD). Intestinal biopsies and anti-CD3/CD28- or anti-CD2/CD28-stimulated lamina propria mononuclear cells from UC and CD patients and control subjects were cultured, and IL-13, IL-4, IL-5, IL-17A and IFN-γ production was measured. Mucosal IL-13-producing cells were characterised by flow cytometry. Gut explants from strictured CD, non-strictured CD and healthy donors were cultured ex vivo, and secreted IL-13, IL-1ß and collagen were measured. IL-13 production by mucosal explants and activated lamina propria mononuclear cells did not differ between CD, UC and control subjects, and was at least a log lower than IFN-γ and IL-17A. IL-13-producing cells, and in particular natural killer T cells, were uniformly low in all groups. IL-4 and IL-5 were undetectable in culture supernatants. Explants of CD strictures produced low amounts of IL-13, whereas IL-1ß and collagen were elevated. We could not confirm that UC or strictured CD are associated with elevated IL-13 production. These data suggest that an anti-IL-13 Ab would not be an appropriate therapeutic strategy in inflammatory bowel disease.

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses.

Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood. Here we compared CD1c(+) myeloid DC (mDC)1, BDCA-3(+)mDC2, and plasmacytoid DC (pDC) in peripheral blood and lymphoid tissues for phenotype, cytokine production, and their capacities to prime cytotoxic T cells. mDC1 were surprisingly the only human DC that secreted high amounts of IL-12p70, but they required combinational Toll-like receptor (TLR) stimulation. mDC2 and pDC produced interferon-λ and interferon-α, respectively. Importantly, mDC1 and mDC2 required different combinations of TLR ligands to cross-present protein antigens to CD8(+) T cells. pDC were inefficient and also expressed lower levels of major histocompatibility complex and co-stimulatory molecules. Nevertheless, all DC induced CD8(+) memory T-cell expansions upon licensing by CD4(+) T cells, and primed naive CD8(+) T cells following appropriate TLR stimulation. However, because mDC1 produced IL-12, they induced the highest levels of cytotoxic molecules. In conclusion, CD1c(+)mDC1 are the relevant source of IL-12 for naive T cells and are fully equipped to cross-prime cytotoxic T-cell responses.

Fine tuning by human CD1e of lipid-specific immune responses.

CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1-lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1-lipid complexes.

Positive modulation of a new reconstructed human gut microbiota by Maitake extract helpfully boosts the intestinal environment in vitro.

The human gut is a complex environment where the microbiota and its metabolites play a crucial role in the maintenance of a healthy state. The aim of the present work is the reconstruction of a new in vitro minimal human gut microbiota resembling the microbe-microbe networking comprising the principal phyla (Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota), to comprehend the intestinal ecosystem complexity. In the reductionist model, we mimicked the administration of Maitake extract as prebiotic and a probiotic formulation (three strains belonging to Lactobacillus and Bifidobacterium genera), evaluating the modulation of strain levels, the release of beneficial metabolites, and their health-promoting effects on human cell lines of the intestinal environment. The administration of Maitake and the selected probiotic strains generated a positive modulation of the in vitro bacterial community by qPCR analyses, evidencing the prominence of beneficial strains (Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis) after 48 hours. The bacterial community growths were associated with the production of metabolites over time through GC-MSD analyses such as lactate, butyrate, and propionate. Their effects on the host were evaluated on cell lines of the intestinal epithelium and the immune system, evidencing positive antioxidant (upregulation of SOD1 and NQO1 genes in HT-29 cell line) and anti-inflammatory effects (production of IL-10 from all the PBMCs). Therefore, the results highlighted a positive modulation induced by the synergic activities of probiotics and Maitake, inducing a tolerogenic microenvironment.

PCGF6 controls murine Tuft cell differentiation via H3K9me2 modification independently of Polycomb repression.

Cell fate is determined by specific transcription programs that are essential for tissue homeostasis and regeneration. The E3-ligases RING1A and B represent the core activity of the Polycomb repressive complex 1 (PRC1) that deposits repressive histone H2AK119 mono-ubiquitination (H2AK119ub1), which is essential for mouse intestinal homeostasis by preserving stem cell functions. However, the specific role of different PRC1 forms, which are defined by the six distinct PCGF1-6 paralogs, remains largely unexplored in vivo. We report that PCGF6 regulates mouse intestinal Tuft cell differentiation independently of H2AK119ub1 deposition. We show that PCGF6 chromatin occupancy expands outside Polycomb repressive domains, associating with unique promoter and distal regulatory elements. This occurs in the absence of RING1A/B and involves MGA-mediated E-BOX recognition and specific H3K9me2 promoter deposition. PCGF6 inactivation induces an epithelial autonomous accumulation of Tuft cells that was not phenocopied by RING1A/B loss. This involves direct PCGF6 association with a Tuft cell differentiation program that identified Polycomb-independent properties of PCGF6 in adult tissues homeostasis.

Microbiota dysbiosis influences immune system and muscle pathophysiology of dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD) is a progressive severe muscle-wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ-free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.

An Intestinal Th17 Subset is Associated with Inflammation in Crohn's Disease and Activated by Adherent-invasive Escherichia coli.

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role.

Reduced humoral response to two doses of COVID-19 vaccine in patients with inflammatory bowel disease: Data from ESCAPE-IBD, an IG-IBD study.

BACKGROUND: Patients on immunosuppressive drugs have been excluded from COVID-19 vaccines trials, creating concerns regarding their efficacy. AIMS: To explore the humoral response to COVID-19 vaccines in patients with inflammatory bowel disease (IBD) METHODS: Effectiveness and Safety of COVID-19 Vaccine in Patients with Inflammatory Bowel Disease (IBD) Treated with Immunomodulatory or Biological Drugs (ESCAPE-IBD) is a prospective, multicentre study promoted by the Italian Group for the study of Inflammatory Bowel Disease. We present data on serological response eight weeks after the second dose of COVID-19 vaccination in IBD patients and healthy controls (HCs). RESULTS: 1076 patients with IBD and 1126 HCs were analyzed. Seropositivity for anti-SARS-CoV-2 IgG was reported for most IBD patients, even if with a lesser rate compared with HCs (92.1% vs. 97.9%; p<0.001). HCs had higher antibody concentrations (median OD 8.72 [IQR 5.2-14-2]) compared to the whole cohort of IBD patients (median OD 1.54 [IQR 0.8-3.6]; p<0.001) and the subgroup of IBD patients (n=280) without any treatment or on aminosalicylates only (median OD 1.72 [IQR 1.0-4.1]; p<0.001). CONCLUSIONS: Although most IBD patients showed seropositivity after COVID-19 vaccines, the magnitude of the humoral response was significantly lower than in HCs. Differently from other studies, these findings seem to be mostly unrelated to the use of immune-modifying treatments (ClinicalTrials.govID:NCT04769258).